Institute of Pathology, Southwest Hospital Affiliated to Third Military Medical University, Chongqing 400038, China.
J Immunol Methods. 2010 Oct 31;362(1-2):60-9. doi: 10.1016/j.jim.2010.09.003. Epub 2010 Sep 15.
To screen and identify two mimic epitopes in the inflammatory site of lipopolysaccharide binding protein (LBP), and to synthesize and purify their corresponding mimic epitope four branched peptide (multiple antigen peptide, MAP).
Using an anti-full length LBP monoclonal antibody as the target molecule, the amino acid sequences of the exogenous peptides were deduced by combining several different techniques including: affinity screening of the phage display peptide library, the lipopolysaccharide (LPS) binding activity assay and competitive inhibition test, cytokine production inhibition, flow cytometry, and DNA sequencing. Basic Local Alignment Search Tool (BLAST) software was used to compare the resulting peptide sequences with the primary structure sequence of the LBP molecule, and thus the amino acid sequences for two mimic inflammatory epitopes for the binding of LBP and LPS were determined. Additionally, the two target sequences were coupled, and the 9-fluorenylmethyloxycarbonyl (FMOC) solid-phase synthesis method was used to synthesize the 24aa peptide. The design program of the multiple antigen peptide (MAP) was used to couple the four tandem peptides with lysine as the core base to produce the branch like structure, and thus, the four branched peptide was synthesized and purified.
Fourteen phage clones (C) with competitive LPS binding activity with LBP were successfully obtained. Among these, the amino acid sequences of the peptides in C2, C19, C57, C77, C85 and C91 showed a homology of more than 90% to the primary structure of LBP. However, the amino acid sequences of C29 and C90, WKAQKRFMKKSG and LKTRKLFWKYKD, respectively, did not show homology to the primary structure of LBP, which were determined to be mimic epitopes of the inflammatory sites in LBP. Further synthesis of the 24aa peptide using FMOC solid-phase synthesis and MAP modification were carried out, the four branched peptide was synthesized and purified, and the purity was found to be higher than 95%. The purified peptide was subjected to mass spectrometry analysis and amino acid analysis, and its molecular weight (3102.77 kDa) and amino acid composition were in accordance with theoretical values.
The amino acid sequence for two mimic epitopes of the inflammatory site of LBP were determined to be WKAQKRFMKKSG and LKTRKLFWKYKD. The MAP was successfully prepared simultaneously and is able to be used as the core antigen protein for the formulation of vaccines. This knowledge will help in future investigations of the functional characteristics of LBP protein, and enhance exploration into new pathways for the prevention and treatment of LPS inflammatory diseases.
筛选和鉴定脂多糖结合蛋白（LBP）炎症部位的两个模拟表位，并合成和纯化相应的模拟表位四分支肽（多抗原肽，MAP）。
以抗全长 LBP 单克隆抗体为靶分子，采用噬菌体展示肽库的亲和筛选、脂多糖（LPS）结合活性测定和竞争抑制试验、细胞因子产生抑制、流式细胞术和 DNA 测序等多种技术相结合，推导出外源性肽的氨基酸序列。使用基本局部比对搜索工具（BLAST）软件将所得肽序列与 LBP 分子的一级结构序列进行比较，从而确定与 LBP 和 LPS 结合的两个模拟炎症表位的氨基酸序列。此外，将两个靶序列偶联，采用 9-芴甲氧羰基（FMOC）固相合成法合成 24aa 肽。采用多抗原肽（MAP）设计程序，以赖氨酸为核心碱基将四个串联肽偶联，产生分支样结构，从而合成并纯化四分支肽。
成功获得了 14 个具有与 LBP 竞争 LPS 结合活性的噬菌体克隆（C）。其中，C2、C19、C57、C77、C85 和 C91 肽的氨基酸序列与 LBP 的一级结构有 90%以上的同源性。然而，C29 和 C90 的氨基酸序列，即 WKAQKRFMKKSG 和 LKTRKLFWKYKD，与 LBP 的一级结构没有同源性，被确定为 LBP 炎症部位的模拟表位。进一步采用 FMOC 固相合成和 MAP 修饰合成 24aa 肽，合成并纯化四分支肽，其纯度高于 95%。对纯化的肽进行质谱分析和氨基酸分析，其分子量（3102.77 kDa）和氨基酸组成与理论值相符。
确定了 LBP 炎症部位的两个模拟表位的氨基酸序列为 WKAQKRFMKKSG 和 LKTRKLFWKYKD。同时成功制备了 MAP，并可作为疫苗制剂的核心抗原蛋白。这一知识将有助于进一步研究 LBP 蛋白的功能特性，并为 LPS 炎症性疾病的防治探索新途径。
